PROJECT SUMMARY Protein biosynthesis directly couples genotype to phenotype in the cell, and its regulation is central to cellular physiology. Our understanding of the molecular mechanism of protein synthesis has undergone a revolution in the last decade, built on rapid advances in structural biology and systems biology. However, important questions relating to dynamic events in translation remain unanswered, as the transient nature of these events makes them difficult to isolate. In this application, we propose to decipher the molecular mechanisms of how eukaryotic translation initiation factor 3 (eIF3) in humans regulates translation initiation. We recently discovered that human eIF3 serves multiple roles in translation. Human eIF3 generally helps assemble translation preinitiation complexes at the start codon, but also directly controls the translation of specific mRNAs. We found that eIF3 can either activate or repress the translation of an mRNA, depending on how eIF3 binds to the mRNA's 5' untranslated region (5' UTR). Furthermore, we discovered that eIF3 harbors a subunit?EIF3D?that binds the 5'-m7G cap on certain mRNAs in an RNA structure-dependent manner. These discoveries indicate that eIF3 may integrate multiple signals to control the translational output of individual mRNAs, much like the Mediator complex in transcription. The aims in this application build on these groundbreaking results to address fundamental questions of how eIF3 functions. We propose to determine the structural basis for eIF3-mediated control of specialized translation, using cryo-electron microscopy (cryo-EM) and in vitro biochemistry. We will also probe how mRNAs regulated by eIF3 are structured in living cells, and dissect the functional importance of these structures to eIF3 regulation and the translational capacity of these mRNAs. Finally, we will use systems biological approaches to explore how eIF3 contributes to the regulation of translation mediated by N-6- methyladenosine (m6A) modifications in mRNAs. By combining advances in cryo-EM to our expertise in human cell engineering, we are in a unique position to unravel the molecular contributions of eIF3 to translation initiation in humans. Taken together, the three aims of this application build on the fundamental insights into eIF3 structure and function obtained in the prior funding period, and address key mechanisms in translational control that could be widespread in human biology. In the long run, our insights into the molecular mechanisms used in human cells to direct eIF3-mediated activation and repression of specific mRNAs could pave the way for the development of new small-molecule and cell-based therapeutics.